For virus research and research and development of antiviral drugs, an experimental system that allows efficient viral amplification is absolutely essential. Further, when there is a system for amplifying virus by the use of cultured cells or a system for evaluating virus proliferation by the use of cultured cells, virus research and research and development of antiviral drugs show dramatic progress.
Hepatitis C virus (HCV) is a virus belonging to the flavivirus family with a single-stranded (+) sense strand RNA as the genome and is known to cause hepatitis C. Recent studies have revealed that the hepatitis C virus can be classified into many types depending on genotypes or serotypes. According to the method of systematic analysis by Simmonds et al. using nucleotide sequences of HCV strains that is currently the mainstream method for classification of HCV genotypes, HCVs are classified into six types, i.e. genotype 1a, genotype 1b, genotype 2a, genotype 2b, genotype 3a, and genotype 3b, and further each type of them is classified into several subtypes (Simmonds, P. et al., Hepatology (1994) 10, p 1321-1324). At present, the full length genome nucleotide sequences of a plurality of HCV genotypes have been determined (Choo et al., Science, (1989) 244, p 359-362; Kato et al., J. Med. Virol., (1992) p 334-339; Okamoto, H et al., J. Gen. Virol., (1992) 73, p 673-679; Yoshioka et al., Hepatology, (1992) 16, 293-299).
Current major treatment for hepatitis C is performed with interferon-α, interferon-β, and a combination therapy of interferon-α and ribabirin that is a purine nucleoside derivative. However, even when these treatments are performed, the therapeutic effect is observed only in about 60% of all patients who underwent therapy, and more than half of the patients who had the effect relapse when the treatment was stopped. The therapeutic effect of interferon is known to be associated with HCV genotypes and it is said that its effect on genotype 1b is low while its effect on genotype 2a is higher (Mori, S., et al., Biochem. Biophys. Res. Commun., (1992) 183, 334-342).
The development of effective therapeutic drug or prophylactic drug for hepatitis C that has high morbidity in industrial countries and finally leads to a serious result and for which there is presently no causal therapy is an important challenge. Therefore, progress in chemotherapy specific to HCV or vaccine therapy and development of anti-HCV drug are eagerly awaited. As a target for the development of anti-HCV drug, suppression of HCV replication and suppression of cell infection of HCV are conceivable.
Since, until recently, it has been difficult to propagate HCV in a cell culture system as well as to infect cultured cells by HCV and animals that can be infected by HCV and subjected to experiments have been limited only to chimpanzee, researches on mechanism of HCV replication and mechanism of HCV infection have remained difficult. However, HCV subgenomic RNA replicons have recently been created as RNAs that have been derived from HCV and have autonomous replication ability (JP Patent Publication (Kokai) No. 2001-17187 (2001); Lomann et al., Science, (1999) 285, 110-113; Blight et al., Science, (2000) 290, 1972-1974; Friebe et al., (2001) 75, 12047-12057; Ikeda et al., J. Virol., (2002) 76, 2997-3006), thereby allowing the mechanism of HCV replication to be analyzed by the use of cultured cells. These HCV subgenomic RNA replicons are ones in which structural proteins that are present downstream of HCV IRES in the 5′ untranslated region of HCV genomic RNA of a clone referred to as Con 1 belonging to genotype 1b are substituted by neomycin-resistant gene and EMCV-IRES linked to the downstream thereof. It has been demonstrated that these replicon RNAs autonomously replicate in Huh7 cells by introducing them into human hepatocarcinoma Huh7 cells and culturing in the presence of neomycin. An evaluation system of HCV replication using this RNA replicon system is considered to become a powerful tool for the development of anti-HCV drug.
It has been reported that, in HCVs different in genotypes, encoded viral proteins also differ, and it is conceivable that full elucidation of the mechanism of HCV replication is difficult only by analyzing the subgenomic RNA replicon derived from genotype 1b HCV. Further, it is assumed that the development of an anti-HCV drug that exerts an effect on various types of HCVs by using only the HCV replication system containing the subgenomic RNA replicon of genotype 1b HCV is particularly difficult because the therapeutic effect of interferon differs depending on HCV genotypes. Accordingly, it is considered that researches on the mechanism of HCV replication and the anti-HCV drug should be conducted by creating HCV RNA replicons of many genotypes.
At present, there are only several lines of clones that can replicate as a replicon in cultured cells. Further, a clone that was cloned from chronic hepatitis and has been confirmed to be infectious to chimpanzees is not necessarily able to replicate as a replicon (Lomann et al., Science, (1999) 285, 110-113). That is, a method for selecting an HCV strain that makes it possible to produce HCV replicon with excellent efficiency of replication as well as with high probability has not yet been found, and when such a selection method of HCV strain is established, the research and development of HCV therapeutic drug is expected to dramatically advance.
For HCV, a vaccine has not been developed at present. One of the reasons for this is that HCV-related proteins that can serve as the vaccine can not be stably produced in a form present in vivo in a large amount. Since HCV-related proteins are expressed in the HCV replicon cells described above (JP Patent Publication (Kokai) No. 2001-17187 (2001)), it is expected that this HCV replicon cells can be used. However, it is necessary to use cells that can be mass-cultured for industrial production of vaccines. From this point of view, Huh7 cells established as the only HCV RNA replicon-replicating cells at present seem to be unsuitable for the vaccine production. For cells suitable for the vaccine production, cells that can be cultured in a very large amount in a suspension culture system such as HeLa cells are conceivable. Furthermore, since the sequence of HCVs differs depending on the genotypes as described above, the vaccine needs to be produced for every genotype. In other words, HCV RNA replicon cells of various genotypes need to be produced using cells suitable for the vaccine production. On the other hand, Huh7 cells are considered not suitable for establishing a cell culture system that allows HCV to autonomously infect and replicate also in the respect that the susceptibility to infection by HCV has not been confirmed. Accordingly, it is thought to be essential to establish a cell culture system that allows HCV to autonomously infect and replicate using other hepatocarcinoma cells.
Moreover, it becomes possible to identify cellular factors necessary for HCV replication by comparing the differences of the mechanism of HCV RNA replicon replication among various kinds of cells, which may be expected to lead to discovery of a novel target for anti-HCV therapeutic drug.